The assembly of functional antigen receptor genes in lymphoid cells (generally known as V (D)j recombination) is known to depend on the products of the RAG-1 and RAG-2 genes, but it has not been clear whether these proteins act directly in recombination or whether they are indirect activators. Recombination is known to begin with a double st rand break in DNA, exactly at the border between each coding segment and its neighboring recombination signal sequence. We have now succeeded in reproducing this specific breakage step in a cell-free reaction requiring only the purified RG-1 and RAG-2 proteins. Thus RAG-1 and RAG-2 act directly in V(D)J recombination, and contain all the information for the site specificity of the reaction.